Effects of Corchorusoside C on NF-κB and PARP-1 Molecular Targets and Toxicity Profile in Zebrafish.

The present study aims to continue the study of corchorusoside C (1), a cardenolide isolated from Streptocaulon juventas, as a potential anticancer agent. A mechanistic study was pursued in a zebrafish model and in DU-145 prostate cancer cells to investigate the selectivity of 1 towards NF-κB and PARP-1 pathway elements. Compound 1 was found to inhibit the expression of IKKα and NF-κB p65 in TNF-α induced zebrafish and inhibit the expression of NIK in vitro. The protein expression levels of XRCC-1 were increased and p53 decreased in DU-145 cells. XIAP protein expression was initially decreased after treatment with 1, followed by an increase in expression at doses higher than the IC50 value. The activity of caspase-1 and the protein expression levels of IL-18 were both decreased following treatment of 1. The binding interactions for 1 to NIK, XRCC-1, p53, XIAP, and caspase-1 proteins were explored in molecular docking studies. Additionally, the toxicity profile of 1 in zebrafish was favorable in comparison to its analog digoxin and other anticancer drugs at the same MTD in zebrafish. Overall, 1 targets the noncanconical NF-κB pathway in vivo and in vitro, and is well tolerated in zebrafish supporting its potential in the treatment of prostate cancer.

A pentamethoxylated flavone from Glycosmis ovoidea promotes apoptosis through the intrinsic pathway and inhibits migration of MCF-7 breast cancer cells.

The rare flavone 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (PMF) has been isolated from several plant species, and its cytotoxic activity has been reported against many types of cancer cells. In this study, PMF was purified from Glycomis ovoidea collected in Vietnam, and its antiproliferative effects and underlying mechanism of action were investigated against MCF-7 cells. PMF inhibited growth in MCF-7 > MCF-10A > MDA-MB-231 cells after 72 hr treatment, with IC50 values of 1.5, 1.9, and 8.6 µg/ml, respectively. Further experiments conducted with this compound in MCF-7 cells, showed the loss of mitochondrial membrane potential, reactive oxygen species overproduction, upregulation of BAX, cytochrome c, caspase-3 and PARP-1 and down-regulation of BCL-2 proteins as well as an increase in caspase-3/-7 activity, suggesting induction of the apoptotic intrinsic pathway. Furthermore, PMF increased cell cycle arrest in the G1 phase, which correlated with increments in the p53 and p21 levels. Additionally, MCF-7 cell migration was inhibited, which could be related to NF-κB p65 downregulation. Finally, PMF did not show toxicity in vivo in a zebrafish (Danio rerio) model. In conclusion, PMF induces cell death in MCF-7 cells through regulation of the BCL-2 protein family and may be proposed as a lead as a potential alternative for breast cancer therapy.

Cytotoxic constituents from Penicillium concentricum, an endophytic fungus from Trichocolea tomentella.

In our continuing effort to identify bioactive secondary metabolites from natural sources, the antiproliferative activity of 23 compounds, previously isolated from Penicillium concentricum, was assessed using the sulforhodamine B assay. The cytotoxic effect was determined against HeLa cervical, HT-29 colon, MDA-MB-321 breast, PC-3, and DU-145 prostate cancer cell lines. Compounds were also tested in the mitochondrial transmembrane potential (MTP) and nuclear factor kappa B (NF-κB) target-based assays. The results showed that 2-bromogentisyl alcohol (2) and 3-hydroxy-benzenemethanol (8) exhibited the highest cytotoxic activity against different cancer cell lines. Epoxydon (14) showed selectivity against DU-145 prostate cancer cells [inhibitory concentration 50 (IC50)=1.2 µmol/l]. Compounds 2, 8, 14, 18, 21 also induced damage of MTP (IC50=0.1, 0.2, 7.0, 9.6, and 1.8 µmol/l, respectively). In the NF-κB assay, only compound 8 exhibited potent inhibition (IC50=0.3 µmol/l). Compounds 2 and 14 showed cytotoxic activity and induction of damage in mitochondrial membrane potential while compound 8 inhibited NF-κB and MTP damage. Additionally, compound 14 with selectivity against DU-145 prostate cancer cells induced cell cycle arrested in G2/M phase. Thus, compounds 2, 8, and 14 could be useful leads in the development of new anticancer agents from natural sources.

Caspase-Dependent Apoptosis in Prostate Cancer Cells and Zebrafish by Corchorusoside C from Streptocaulon juventas.

Corchorusoside C (1), isolated from Streptocaulon juventas collected in Vietnam, was found to be nontoxic in a zebrafish ( Danio rerio) model and to induce cytotoxicity in several cancer cell lines with notable selective activity against prostate DU-145 cancer cells (IC50 0.08 µM). Moreover, corchorusoside C induced DU-145 cell shrinkage and cell detachment. In CCD-112CoN colon normal cells, 1 showed significantly reduced cytotoxic activity (IC50 2.3 µM). A preliminary mechanistic study indicated that 1 inhibits activity and protein expression of NF-κB (p50 and p65), IKK (α and ß), and ICAM-1 in DU-145 cells. ROS concentrations increased at 5 h post-treatment, and MTP decreased in a dose-dependent manner. Moreover, decreased protein expression of Bcl-2 and increased expression of PARP-1 was observed. Furthermore, corchorusoside C increased both the activity and protein levels of caspases 3 and 7. Additionally, 1 induced sub-G1 population increase of DU-145 cells and modulated caspases in zebrafish with nondifferential morphological effects. Therefore, corchorusoside C (1) induces apoptosis in DU-145 cells and targets the same pathways both in vitro and in vivo in zebrafish. Thus, the use of zebrafish assays seems worthy of wider application than is currently employed for the evaluation of potential anticancer agents of natural origin.

Structurally Modified Cyclopenta[b]benzofuran Analogues Isolated from Aglaia perviridis.

Four new cyclopenta[b]benzofuran derivatives based on an unprecedented carbon skeleton (1-4), with a dihydrofuran ring fused to dioxanyl and aryl rings, along with a new structural analogue (5) of 5‴-episilvestrol (episilvestrol, 7), were isolated from an aqueous extract of a large-scale re-collection of the roots of Aglaia perviridis collected in Vietnam. Compound 5 demonstrated mutarotation in solution due to the presence of a hydroxy group at C-2‴, leading to the isolation of a racemic mixture, despite being purified on a chiral-phase HPLC column. Silvestrol (6) and episilvestrol (7) were isolated from the most potently cytotoxic chloroform subfraction of the roots. All new structures were elucidated using 1D and 2D NMR, HRESIMS, IR, UV, and ECD spectroscopic data. Of the five newly isolated compounds, only compound 5 exhibited cytotoxic activity against a human colon cancer (HT-29) and human prostate cancer cell line (PC-3), with IC50 values of 2.3 µM in both cases. The isolated compounds (1-5) double the number of dioxanyl ring-containing rocaglate analogues reported to date from Aglaia species and present additional information on the structural requirements for cancer cell line cytotoxicity within this compound class.

α-Pyrone and Sterol Constituents of Penicillium aurantiacobrunneum, a Fungal Associate of the Lichen Niebla homalea.

Four new metabolites, 4-epi-citreoviridin (1), auransterol (3), and two analogues (2 and 4) of paxisterol (6), together with two known metabolites (15R*,20S*)-dihydroxyepisterol (5) and (6), were isolated from cultures of the fungal associate, Penicillium aurantiacobrunneum, of the lichen Niebla homalea, endemic to California and Baja California. The structures of all compounds were determined by comprehensive spectroscopic and spectrometric methods, as well as single-crystal X-ray diffraction for the determination of the absolute configuration of 3. Compound 1 showed selective cytotoxicity toward MCF-7 breast and A2780 ovarian cells with IC50 values of 4.2 and 5.7 µM, respectively.

Cytotoxic and NF-κB and mitochondrial transmembrane potential inhibitory pentacyclic triterpenoids from Syzygium corticosum and their semi-synthetic derivatives.

Syzygium is a large genus of flowering plants, with several species, including the clove tree, used as important resources in the food and pharmaceutical industries. In our continuing search for anticancer agents from higher plants, a chloroform extract of the leaves and twigs of Syzygium corticosum collected in Vietnam was found to be active toward the HT-29 human colon cancer cell line. Separation of this extract guided by HT-29 cells and nuclear factor-kappa B (NF-κB) inhibition yielded 19 known natural products, including seven triterpenoids, three ellagic acid derivatives, two methylated flavonoids, a cyclohexanone, four megastigmanes, a small lactone, and an aromatic aldehyde. The full stereochemistry of (+)-fouquierol (2) was defined for the first time. Biological investigations showed that (+)-ursolic acid (1) is the major cytotoxic component of S. corticosum, which exhibited also potent activities in the NF-κB and mitochondrial transmembrane potential (MTP) inhibition assays conducted, with IC50 values of 31 nM and 3.5 µM, respectively. Several analogues of (+)-ursolic acid (1) were synthesized, and a preliminary structure-activity relationship (SAR) study indicated that the C-3 hydroxy and C-28 carboxylic acid groups and 19,20-dimethyl substitution are all essential in the mediation of the bioactivities observed for this triterpenoid.

Secondary metabolites from Garcinia daedalanthera Pierre leaves (Clusiaceae).

Two new glycerol esters, (S)-2-hydroxy-3-(octanoyloxy)propyl tetracosanoate (1) and (S)-3-(((S)-11-acetoxy octadecanoyl)oxy)propane-1,2-diyl diacetate (2), and eight known compounds, docosanedioic acid (3), 2,5-dimethylnonadecane (4), lupeol (5), stigmasterol (6), ß-sitosterol (7), heptadecanoic acid (8), hexanedioic acid, 1,6-bis[(2R)-ethylhexyl] ester (9), and 1,3-di-O-[2',2'-di-(p-phenylene)] (10) were isolated from the leaves of Garcinia daedalanthera Pierre, collected from Indonesia. Structural analysis of the isolates was performed using 1 D- and 2 D-NMR, LC- and GC-MS, IR, polarimetry, and UV-visible spectroscopic methods. Cytotoxicity assessments, as well as reactive oxygen species (ROS) analysis of the isolates, were also completed. Lupeol was the only compound found active with an IC50 value of 19.2 µM against HT-29 colon cancer cells. Significant ROS inhibition and induction activity was observed for compounds 4 and 8, respectively.

Cytotoxic constituents of Glycosmis ovoidea collected in Vietnam.

Compounds derived from natural sources have been major contributors to the area of cancer chemotherapy for decades. As part of an ongoing effort to discover anticancer drug leads from tropical plants, a large-scale collection of Glycosmis ovoidea Pierre (Rutaceae), was made at Nui Chua National Park, Vietnam. Activity-guided fractionation of the chloroform-soluble fractions led to the isolation of nine coumarins, including the new compound, 1-(7-methoxy-2-oxo-2H-chromen-8-yl)-3-methyl-1-oxobut-2-en-2-yl (S)-2-methylbutanoate (1). An close analogue of 1, namely, kincuongin (2), was deemed as non-cytotoxic (IC50 > 10 µM) against five different cancer cell lines. However, co-administration of kimcuongin (2) showed an approximately 100 times potentiation of the MCF-7 breast cancer cell cytotoxicity of the previously reported flavonoid, 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (10). To provide a mechanistic basis for the cancer cell line inhibition enhancement observed, an initial in silico study on compound 10 indicated that it interacts with isoforms of the NF-κB complex. In a confirmatory western blot experiment conducted, kimcuongin (2) was found to potentiate the effects of flavone 10 in inhibiting both NF-κB and PARP-1. In vivo investigations using a zebrafish (Danio rerio) model showed that compounds 2, 3, 5, and 6 did not exhibit any discernible toxicity at concentrations up to 50 µM.

Spermidine alkaloid and glycosidic constituents of Vietnamese Homalium cochinchinensis.

Phytochemical investigation of the aerial parts of Homalium cochinchinensis led to the isolation of secondary metabolites belonging to the spermidine alkaloid, glycoside, depsidone and phenol classes. Of the eleven secondary metabolites isolated in this study, two spermidine alkaloids, dovyalicins H (1) and I (2), which belong to a rare group among this class, and six glycosides (3-8) are previously undescribed. The structures of all new isolates were determined by interpretation of spectroscopic and spectrometric data. In this report, the structural elucidation of these unprecedented secondary metabolites (1-8) is described.

Apoptosis Induced by (+)-Betulin Through NF-κB Inhibition in MDA-MB-231 Breast Cancer Cells.

BACKGROUND/AIM: This study aimed to uncover the effects of (+)-betulin on the NF-κB-apoptotic pathway in MDA-MB-231 cells, and determine its toxicity and protein expression in vivo. MATERIALS AND METHODS: Cell cytotoxicity and toxicity were determined using the SRB assay and a zebrafish model, respectively. Western blot, mitochondrial transmembrane potential (MTP), and computational modeling analysis were performed. RESULTS: (+)-betulin inhibited the growth of MDA-MB-231 cells, but did not induce toxicity in zebrafish. (+)-Betulin inhibited the activity of NF-κB p65 in silico and in vitro. In cells, (+)-betulin down-regulated NF-κB p50 and 65, IKKα and ß, ICAM-1 and bcl-2 expressions. Cell co-treatment with (+)-betulin and TNFα increased the (+)-betulin cytotoxic potential. Moreover, (+)-betulin induced the loss of MTP. Furthermore, (+)-betulin, in zebrafish, down-regulated the expression of NF-κB p65, IKKα, ΙΚΚß and procaspase-3. CONCLUSION: The results contribute to the understanding of the mode of action on apoptosis induction by inhibiting NF-κB pathway in MDA-MB-231 cells.

Tumor suppressor p53 independent apoptosis in HT-29 cells by auransterol from Penicillium aurantiacobrunneum.

Colorectal cancer is the third leading cause of cancer related-death in the United States. Search for new alternatives to treat this type of cancer is necessary. In a previous report, auransterol from Penicillium aurantiacobrunneum showed cytotoxicity in HT-29 cancer cells. Thus, the goal of this study was to examine the potential cytotoxic mechanism of auransterol in HT-29 cells. Real-time cytotoxicity of auransterol was determined in HT-29 colon cancer cells, using the SRB assay. Loss of MTP, overproduction of ROS, cell cycle, cell migration, and caspase activity were analyzed. Western blot analysis was used to evaluate protein expression. Auransterol reduced cell proliferation rate in a time and concentration-dependent manner, with an IC50 value > 100, 49.1 and 23.8 µM at 24, 48 and 72 h of treatment, respectively. After 24 h of treatment, 50 µM of auransterol induced loss of MTP, overproduction of ROS, increased caspase activity, induced cell cycle G1 phase accumulation and inhibition of migration in HT-29 cells compared to control. These results were supported by protein upregulation of Cyt c, BAX, PARP-1, p21 and procaspase-3, and downregulation of Bcl-2 with no modifications in procaspase-7 and p53. The cytotoxic effect of auransterol in HT-29 colon cancer cells is mediated by mitochondrial apoptosis independent of p53 activation, cell cycle G1 phase arrest, and inhibition of cell migration. This work encourages further preclinical and clinical studies of auransterol and suggests auransterol as a good candidate for colorectal cancer treatment.

Apoptotic activity of xanthoquinodin JBIR-99, from Parengyodontium album MEXU 30054, in PC-3 human prostate cancer cells.

Natural products are a valuable source of anticancer agents, with many naturally derived compounds currently used in clinical and preclinical treatments. This study aims to investigate the antiproliferative activity and potential mechanism of action of the xanthoquinodin JBIR-99, isolated from fungi Parengyodontium album MEXU 30,054 and identified by single-crystal X-ray crystallography. Cytotoxicity of xanthoquinodin was evaluated in a panel of human cancer cells lines and CCD-112-CoN normal colon cells, using the sulforhodamine B assay. PC-3 prostate cancer cells were used in biochemical assays including cell cycle, mitochondrial transmembrane potential (MTP), reactive oxygen species (ROS) and caspase activity. Expression levels of apoptosis-pathway-related proteins were analyzed by Western blot. The in vivo toxicity of xanthoquinodin was determined using a zebrafish model. Xanthoquinodin showed cytotoxicity in all cancer cell lines but demonstrated relative selective potency against PC-3 cells with an IC50 1.7 µM. In CCD-112-CoN cells, xanthoquinodin was non-cytotoxic at 100 µM. In PC-3 cells, the compound induced loss of MTP, production of ROS, and cell cycle arrest in S phase. The expression and activity of caspase-3 was increased, which correlates with the upregulation of Cyt c, Bax, nuclear factor kappa-B (NF-κB) (p65) and IKKß, and downregulation of poly ADP ribose polymerase (PARP-1) and Bcl-2. Lastly, xanthoquinodin did not cause any visible developmental toxicity in zebrafish at 50 µM. These results demonstrate xanthoquinodin induces apoptosis in PC-3 prostate cancer cells by activation of both intrinsic and extrinsic apoptotic pathways. In addition, the non-toxic effect in vivo indicates that xanthoquinodin could be a useful lead in the development of a novel, anti-cancer agent that is selective for prostate cancer.

Antinociceptive activity of the essential oil from Artemisia ludoviciana.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Artemisia ludoviciana are widely used in Mexico for treating gastrointestinal disorders, painful complaints and diabetes. AIM OF THE STUDY: To establish the preclinical efficacy as antinociceptive agent of the essential oil (EO) from the aerial parts of A. ludoviciana using well-known animal models. MATERIALS AND METHODS: Acute antinociceptive effect of EO (1, 10, 31.6, 100, and 316mg/kg, i.p.) was evaluated using the hot plate and paw formalin models in mice. The motor effects were assessed with the rota-rod and open field assays. The volatile components obtained by headspace solid phase microextraction (HS-SPME) and hydrodistillation were determined using gas chromatography coupled with mass spectrometry (GC-MS) analysis. RESULTS: EO decreased first and second phases of formalin test; in the first stage, the better effect was obtained with the treatment of 316mg/kg but in the second phase, time licking was attenuated at the doses of 31.6, 100 and 316mg/kg. The effectiveness of EO (ED50=25.9mg/kg) for attenuating neurogenic pain was corroborated using the hot plate test. The antinociceptive action of EO was blocked by naloxone suggesting that its mode of action involved an opioid mechanism. Furthermore, EO (316mg/kg) did not affect animal motor and coordination functions when tested by the rota-rod and open field tests. The latter results indicated that the pharmacological effects exerted by EO during the hot plate and formalin test are truly antinociceptive. GC-MS analysis of EO revealed that (±)-camphor, γ-terpineol, 1,8-cineole and borneol were the major volatile compounds of the plant. CONCLUSION: EO from A. ludoviciana showed significant antinociceptive effect, which appeared to be partially mediated by the opioid system. These findings could support the long-term use of A. ludoviciana for treating painful complaints in Mexican folk medicine.

Hypoglycemic properties of some preparations and compounds from Artemisia ludoviciana Nutt.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia ludoviciana, commonly known as "estafiate", plays an important role in contemporary Mexico for treating several diseases including diabetes. To establish the preclinical efficacy of Artemisia ludoviciana as hypoglycemic and/or antihyperglycemic agent using well-known animal models. MATERIALS AND METHODS: Acute hypoglycemic as well as oral glucose (OGTT) and sucrose (OSTT) tolerance tests were used to demonstrate the value of the plant for treating diabetes. An essential oil (EO), an organic extract (OE) and an infusion (AE) were assayed in normal and NA-STZ-treated mice. The acute toxicity of the three preparations was analyzed by the Lorke method. The infusion was subjected to conventional phytochemical study using chromatographic conventional procedures. Some of the isolates were evaluated using the same pharmacological assays as well as an enzymatic test. The latter was employed to assess their potential inhibitory effect on yeast α-glucosidase. RESULTS: Oral administration of OE to normal mice significantly decreased blood glucose level only at the dose of 100 mg/kg; in NA-STZ-mice the hypoglycemic effect was observed at the three doses tested (31.6, 100, and 316 mg/kg). The infusion reduced significantly, blood sugar levels only in diabetic mice; the best effect was observed at the dose of 316 mg/kg. The EO was inactive when evaluated in normal mice. Regarding to the antihyperglycemic effect, the best effect was observed with the OE, during the OGTT and OSTT in diabetic mice. The infusion (AE) showed better effects during the OGTT in both normal and diabetic animals at the dose of 100 mg/kg. Finally, the EO was inactive during an OGTT at the three doses tested (31.6, 100, and 316 mg/kg) in diabetic mice. In addition, the results of AE on the enzymatic test using yeast α-glucosidase revealed an inhibition of 45%; this finding was consistent with the results obtained with the same preparation in vivo during an OSTT. Conventional phytochemical analysis of the active AE led to the isolation and characterization of eupatilin (1), jaceosidin (2), arglanin (3), salvinine (4), and 3,5-dicaffeoylquinic acid (5). Biological testing of 1 and 3 revealed their hypoglycemic effect. The hypoglycemic effect of arglanin (3) was attenuated in the presence of nicorandil, which suggested that the lactone behaved as an ATP-K+-channel blocker as glibenclamide. Salvinine (4) turned out to be a mixed α-glucosidase inhibitor, while 3 was inactive. CONCLUSIONS: Artemisia ludoviciana preparations showed hypoglycemic and antihyperglycemic effects, which could explain its effectiveness for treating diabetes in contemporary Mexico. Some of the active principles of the plant included compounds 1-5. These compounds seem to be acting synergistically on different molecular targets which involved glucose absorption and insulin liberation.